PROJECT SUMMARY The role of the bi-functional Chlamydia trachomatis protein, Scc4 (formerly Ct663), in regulating the type III secretion system (T3SS) and the essential, developmental cycle (between infectious elementary bod- ies and actively dividing, reticulate bodies) is not well-understood at the molecular level. Scc4 is a unique protein that functions (i) as a T3SS chaperone with Scc1 to regulate secretion of CopN (an essential viru- lence factor) and (ii) as a transcription factor regulating ?66-RNA polymerase holoenzyme. Based on pro- gress made during the last funding period, the switching mechanism proposed for Scc4?s dual functions involves a conformational change in its 3-dimensional structure. Because Scc4 has two essential func- tions and is unique to C. trachomatis, it is a novel target for developing anti-virulence drugs. In order to develop drugs targeting the T3SS and developmental cycle of C. trachomatis, the structural mechanisms controlling Scc4?s chaperone function and its interaction with its protein partners must first be deter- mined. The long-term goal of the proposed research is to screen and design drugs targeting Scc4?s func- tions, which would prevent the bacteria from spreading, likely curing C. trachomatis directly and certainly mitigating the development of antibiotic-resistant strains, when combined with current therapies. The overall objectives of this application are to understand the switching mechanism of Scc4 between its free and chaperone-complex forms. The central hypothesis is that a conformational switch in Scc4?s structure regulates its dual activity. The rationale for the proposed research is that inhibitors to disrupt Scc4?s chaperone functions can be designed, resulting in new approaches to treat C. trachomatis and new mo- lecular tools to study the T3SS and developmental cycle. The central hypothesis and objectives of this application will be tested and attained by pursuing two specific aims: determine the high-resolution 3D structure of (1) Scc4 and (2) Scc4 in complex with Scc1. It is anticipated that these aims will yield the ex- pected outcome of a structural model of Scc4?s switching mechanism for rational drug design. This out- come is expected to have an important positive impact because structures of Scc4 will provide the infor- mation necessary to develop drugs targeting Scc4?s conformational switch and/or binding interface with Scc1 fundamentally advancing the fields of structural biology and infectious disease biology. This contri- bution is significant because it is the first step in a continuum of research that is expected to lead to un- derstanding Scc4?s dual functions at the atomic level and contribute to the treatment of the most com- mon, sexually transmitted bacterial disease. The proposed research is innovative because the NMR structural and biochemical characterization of a unique bi-functional protein from C. trachomatis to target bacterial virulence as a therapeutic strategy represents a new and substantive departure from the status quo.